International standards: At present, the traditional methods still apply to international standards in detecting low numbers of pathogens in food samples. Many standard methods include a two-stage enrichment culture. The first step, or pre-enrichment, involves suspending the required sample weight (10-25g) in a large (100-250ml) volume of a non-selective medium designed to resuscitate damaged cells and promote microbial growth. After incubation, typically at 37oC for 18-24 hours, a small volume of the culture is transferred to a suitable selective enrichment medium designed to suppress the growth of competing organisms while allowing the target pathogen to multiply. In theory, such methods can amplify a single viable cell to detectable levels, even if it remains attached to the food matrix. There is no need to treat the sample to ensure that microbial cells are freely suspended in the medium. Though simple and reliable, traditional enrichment culture-based methods are extremely time-consuming, often taking several days to complete.

Food sample suspensions

Pathogen detection methods that do not utilize some form of enrichment culture still require the preparation of a sample suspension before separation, concentration, and detection techniques can be applied. Ideally, the initial suspension will be prepared in such a way that each target cell present is freely suspended in the diluent. Most foods will require a homogenization step using a stomacher or a blender. Blenders should not be used in measuring A_w as they can generate heat, causing water present in the food to evaporate and effect A_w readings.

Traditional quantitative methods (e.g., for total viable counts) often specify some form of mechanical agitation to release microbial cells from the sample matrix. Some protocols still include rotary bladed blenders as the preferred method of producing a suspension, but the need to clean and sterilize the blender between samples and problems with heat generation in the suspension limit their effectiveness. Paddle type blenders like the Seward Stomacher® overcome some of these problems by processing the sample in a sterile plastic bag so that the paddles do not make contact with the sample and remain clean. They are also able to handle large volumes of liquid. Paddle blenders are now widely used in food microbiology laboratories and have replaced bladed blenders.

A more recent development is the Pulsifier® from Microgen Bioproducts, which replaces the paddles with a high-speed oscillating ring, producing shock waves as well as agitation. The Pulsifier can release more microbial cells into suspension with less disruption to the food matrix than other blenders, and without creating large quantities of food debris in the suspension. Other methods for preparing initial suspensions, such as sonication, have been investigated, but are not widely used.

Separation and concentration

To note: Once microbial cells have been released from the food matrix into suspension, the target pathogen can be separated, and the cells concentrated for more rapid detection. Many different physical, chemical, and immunological methods have been devised to accomplish this, including ion exchange columns and biphasic partitioning. But three techniques form the basis of most practical applications: membrane filtration, centrifugation, and immunomagnetic separation (IMS).

1. Membrane filtration

Filtration has obvious attractions as a means of separating and concentrating microbial cells from sample suspensions. It is relatively inexpensive and straightforward, with the potential to process large volumes of suspension quickly. Some filtration products on the market can work while the food is being homogenized. This is usually called a pre-filtration step and allows a more advanced filtration membrane to work more efficiently. The InnovaPrep Concentrating Pipette, for instance, will automatically run the diluent through its filtration membrane of which microbes attach to. An elution is produced using proprietary wet foam technology and can now be lysed and run on a PCR thermocycler to fulfill pathogen DNA detection requirements. This whole process, which also includes the enrichment stage, takes only 8 hours.

There are difficulties involved when working with high-fat processed foods when filtering. Food debris can block the membrane and prevent some of the target cells being collected. Some improvements in the filterability of food suspensions can be achieved by techniques such as trypsin digestion or the addition of surfactants, but this may also interfere with pathogen detection and reduce cell viability.

For filterable samples, such as beverages and some dairy products, practical separation and concentration methods based on membrane filtration have been developed and combined with a variety of microbial detection technologies as commercially available instruments and test systems. Examples include the ScanRDI from BioMeriux, which uses a solid-phase cytometry technique to detect viable cells directly on filters. Microscopy-based methods, such as the direct epifluorescent filter technique (DEFT), are also still used in some laboratories to count bacterial cells on membrane filters.

2. Centrifugation

Centrifugation has also been used successfully to separate and concentrate microbial cells in food suspensions, although applications are limited by the practical considerations of processing large volumes of liquid. Low-speed centrifugation can be used to remove food debris while leaving bacterial cells in suspension, and higher speeds can then be used to concentrate bacteria. Alternatively, methods based on density gradient centrifugation can be used. For example, buoyant density centrifugation (BDC) has been used in rapid detection of foodborne pathogens. It separates bacterial cells and food particles with different buoyant densities by flotation and sedimentation and to remove PCR inhibitors. Some commercial applications have also been developed, notably the automated Foss Bactoscan™ FC instrument designed for the dairy industry. This can separate bacterial cells from milk with the aid of a density gradient, before detection and enumeration by flow cytometry.

3. Immunomagnetic separation (IMS)

IMS technology was first developed in the 1980s and is now widely used in food microbiology to separate target pathogens from other microorganisms. Magnetized polystyrene beads or other particles are coated with antibodies specific to the target organism, and these are then added to a volume of the sample directly (where high counts are likely), or more often to a pre-enrichment culture. After a short incubation, the target cells are immobilized on the surface of the beads and can be concentrated into a pellet using a powerful magnetic field. The sample can then be discarded, and the pellet is resuspended and washed to remove food debris and other material. The immobilized pathogens can be detected by culture, or by rapid methods, including PCR and ELISA. IMS is a very effective separation and concentration technique, although it is not suitable for processing large volumes of sample or culture, partly because high concentrations of beads are needed to bind the target cells.

Magnetic beads and particles for food microbiology applications have been available commercially for many years. The best known are Dynabeads®, supplied by ThermoFisher Scientific and designed for selective capture and concentration of a range of foodborne pathogens from pre-enrichment cultures. Dynabeads specific to Salmonella Listeria and several VTEC serotypes are available. Dynabeads can also be used in an automated process using the BeadRetriever™ system. Further development of automated IMS technology is illustrated by the Pathatrix® Auto System, also from Thermo Fisher. Pathatrix is unique in allowing up to ten samples to be pooled after pre-enrichment, providing considerable cost savings through reduced detection testing. The separation and concentration process can be completed within 15 minutes.

Future developments

Although methods and instruments designed to speed up the process of separating and concentrating microorganisms from foods are available, there is still a need for new methods to be developed to support PCR and immunology-based detection tests. At present food microbiologists are still reliant on enrichment cultures to amplify low numbers of pathogens to a detectable level, and the goal of instant detection has yet to be achieved.

One approach to moving away from dependence on enrichment procedures is to combine some of the technologies described above in multi-step methods. For example, a protocol using a combination of filtration, low and high-speed centrifugation and buoyant density gradient centrifugation has been described. It is capable of a 250-fold concentration of target pathogens in food samples and detected 10 CFU/g of Salmonella in chicken in 3 hours when combined with a PCR detection technique. Such a method would not be practical for routine application in a food microbiology laboratory but does indicate the potential for the development of multi-step methods, especially if they can be automated.

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